1. Field of the Invention
The present invention relates to transgenic animals as urinary bioreactors for the expression and production of polypeptides in the urine. The present invention further relates to a recombinant DNA construct for kidney-specific expression of polypeptides in the urine and to a method for producing such polypeptides in the urine.
2. Description of the Related Art
Significant progress has recently been made in using transgenic animals as bioreactors to produce large quantity and high quality pharmaceuticals. The overall strategy entails the use of tissue-specific promoters to drive the expression of genes encoding medically important molecules. When those molecules are expressed in the target tissue of transgenic animals and secreted into body fluids, they can be harvested, purified and used for treating human diseases. The most notable example is the milk-based bioreactor system, taking advantage of mammary gland-specific gene promoters. U.S. Pat. No. 5,476,995 was one of the first patents directed to transgenic female sheep as milk-based bioreactors that expressed the transgene in the mammary gland so as to produce the target protein in its milk.
A number of proteins have been produced in milk-based bioreactor systems, such as protein C (U.S. Pat. No. 5,589,604), blood coagulation factors (U.S. Pat. No. 5,322,775), fibrinogen (U.S. Pat. No. 5,639,940), antibodies (U.S. Pat. No. 5,625,126) and hemoglobin (U.S. Pat. No. 5,602,306), some of which are now being used in clinical trials. However, even in view of its initial success, a milk-based bioreactor system has several limitations. The first relates to its relatively low degree of cost-effectiveness. For instance, the lactation of transgenic livestock does not occur until an average of one and a half years old. Besides, lactation only occurs in female animals and lasts for a limited period of time. Secondly, purification of target proteins from milk often requires the development of complicated purification schemes (Wilkins et al, 1992). Thirdly, leakage of biologically active proteins from the mammary gland into the blood stream commonly occurs with the possibility of leading to pathological conditions in transgenic animals.
Another potential bioreactor system that can circumvent some of the above-mentioned limitations is a urine-based system where urine is an easily collectable fluid from transgenic livestock animals. This bioreactor system has been recently tested by Kerr and colleagues (1998), among whom is one of the present inventors, in transgenic mice using a urothelium-specific promoter (uroplakin II promoter) to drive human growth hormone (hGH) expression and production. They found that hGH could indeed be found in the urine of these transgenic mice at a concentration of 0.1 mg/ml, indicating that the urothelium can serve as an alternate bioreactor. The major advantages of this urine-based system over milk-based systems are the ability to harvest the product soon after birth and throughout the life of the animal irrespective of sex or reproductive status and the ease of product purification from urine. In addition, livestock urine is a proven, currently utilized source of pharmaceuticals; it is estimated that urine is being collected from 75,000 pregnant horses annually as a source of estrogenic compounds for postmenopausal hormone replacement therapy (Williams, 1994).
Despite these major advantages, several technical problems still exist with the above-mentioned urine-based bioreactor system, the most important being the relatively low yield of urinary hGH (0.1 mg/ml) obtained by Kerr et al (1998), as most of the hGH appear to be trapped in the cytoplasm of the superficial urothelial cells. This relatively low yield may be because the urothelium is not known to be a major secretory epithelium and the purification of a minor protein from urine may require sophisticated purification procedures. In addition, low levels of hGH was found to have leaked into the mouse blood stream, possibly being responsible for the infertility observed in the transgenic female mice.
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